Natural oxidase-mimicking copper-organic frameworks for targeted identification of ascorbate in sensitive sweat sensing

Sweat sensors play a significant role in personalized healthcare by dynamically monitoring biochemical markers to detect individual physiological status. The specific response to the target biomolecules usually depends on natural oxidase, but it is susceptible to external interference. In this work, we report tryptophan- and histidine-treated copper metal-organic frameworks (Cu-MOFs). This amino-functionalized copper-organic framework shows highly selective activity for ascorbate oxidation and can serve as an efficient ascorbate oxidase-mimicking material in sensitive sweat sensors. Experiments and calculation results elucidate that the introduced tryptophan/histidine fundamentally regulates the adsorption behaviors of biomolecules, enabling ascorbate to be selectively captured from complex sweat and further efficiently electrooxidized. This work provides not only a paradigm for specifically sweat sensing but also a significant understanding of natural oxidase-inspired MOF nanoenzymes for sensing technologies and beyond.

The morphologies and structures of the materials were examined by field-emission scanning electron microscopy (FSEM, Nova Nano SEM 450) and transmission electron microscopy (TEM: Talosf200s).
The Fourier transform infrared spectra of samples for amino acid functionalization were examined by an attenuated total reflectance infrared spectrophotometer (Nicolet iS5) and atomic force microscopybased infrared spectrophotometer (AFM-IR). The Trp/His loading amounts were measured by liquid chromatograph mass spectrometer (LC-MS) (Ultimate 3000 UHPLC-Q Exactive) using methanol as the mobile phase . In situ electrochemical infrared spectroscopy (in situ EC-IR) of the S-3/S-44 samples was recorded by a Fourier transform infrared spectrometer (Nicolet iS50R). The binding behavior of the samples was tested by isothermal titration calorimetry (ITC) (iTC200iTC200) at 25 °C .
The electrooxidation product ascorbate was examined with high-resolution mass spectrometry (HRMS, microTOF Ⅱ ). Electrochemical performances were tested by an electrochemical workstation (CHI760E).

Electrochemical measurement
The electrochemical performance test was performed with a three-electrode system: carbon paper supported STAM-17-OEt or HT-STAM-17-OEt as the working electrode, Ag/AgCl as the reference electrode, and Pt as the counter electrode. The carbon paper electrode area is 0.23758 cm -2 . HT-STAM-17-OEt (4 mg mL -1 ) was dispersed in 200 μL of H 2 O as the ink for coating the carbon paper electrode.
For the human sweat sensing platform, polytetrafluoroethylene (PTFE) was used as the flexible substrate to support the three-electrode system. The concentration of interferents used in the experiment refers to its maximum concentration in human sweat.

HT-STAM-17-OEt-based electrochemical sweat sensing platform
The preparation procedure of the electrochemical sensing platform device is as follows. Polyethylene terephthalate (PET) as the substrate (3 cm×5 cm) was selected for supporting electrodes. Carbon paper electrodes were tailored and pasted on the PET. The polyvinyl butyral (PVB) polymer reference electrode was fabricated according to the reported work.
[1] PVB (400 mg), AgCl (20 mg), and KCl (20 mg) were placed in 5 ml methanol to obtain a homogeneous solution. After that, the mixture was exposed to a light source for 5 min to induce a partial reduction of AgCl. The mixture (500 μL) was dripped on a circular carbon electrode (diameter of 5.5 mm) and dried. HT-STAM-17-OEt and Pt were dripped and fixed on the circular carbon electrode. After drying all the electrodes and materials, the sensing device is completed. The cost of the sweat sensor was evaluated according to the major electrode material cost. The costs of the MOF ligand, AgCl reference and Pt counter electrode are 1300, 350, and 1200 RMB/g, respectively. The masses of ligand, AgCl, and Pt for the sweat sensor are 0.6 mg, 2 mg and 5 mg, respectively. Thus, the cost of a single sweat sensor is 7.5 RMB (0.8 RMB for HT-S-4/S-44 STAM-17-OEt, 0.7 RMB for Ag/AgCl, 6 RMB for Pt). The first author, Zhengyun Wang, as a volunteer, was recruited for collecting sweat in the study, and informed consent was obtained from the individual.

Density functional theory calculation
First-principles calculations were performed within the framework of density functional theory (DFT).
The linear combination of the atomic orbital method and generalized gradient approximation GGA/PW91 functional were employed. Double numerical plus polarisation (DNP) was employed as the basis set. The core electrons were obtained using the all-electron method considering all electrons in the system. The DFT+D method within the OBS scheme was adopted owing to the van der Waals (vdW) weak interaction. The self-consistent field (SCF) tolerance was set as 1×10 -5 Hartree. For geometric optimization, the DFT calculations were performed at a spin-unrestricted set. The amino acids and Cu paddle wheel atoms were free, and the ligand atoms were constrained.

Finite element analysis
The secondary current distribution was used as the physical field to simulate the current density distribution on the surface of the micro/nanostructures of STAM-17-OEt and HT-STAM-17-OEt. To simplify the finite element analysis for micro/nanostructures, the geometry of the electrolyte was abstracted as a cylinder (radius: 20 µm, height: 10 µm), which completely includes the cuboid. The cylinder surface was set as the cathodic electrode, and the 3D cuboid surface was set as the anodic electrode. The electrode kinetic equations for cathodic and anodic electrodes are the Butler-Volmer equations and concentration-dependent Butler-Volmer equations, respectively. The boundary condition parameters for Figure 5G and Figure 5H are included in Table S3. S-5/S-44 To achieve Cu 2 (OH) 3 Cl, CaCO 3 paper can react with H +, which is produced by the hydrolysis of CuCl 2 .
The slow hydrolysis reaction process is as follows: 2Cu 2+ +Cl -+3H 2 O Cu 2 (OH) 3 Cl+3H + s-1 Interestingly, we discovered that using CuCl 2 aqueous solutions with different concentrations would lead to different structures of Cu 2 (OH) 3 Cl on CaCO 3 paper ( Figure S1 and S2). When the concentration is relatively high (1 M), the obtained Cu 2 (OH) 3 Cl shows a small size ( Figure S1A). When the concentration of the CuCl 2 aqueous solution is relatively low (0.2, 0.1 and 0.05 M), the obtained Cu 2 (OH) 3 Cl particles show larger and irregular sizes (Figure S1b-S1d). When the concentration is moderate (0.5 M), Cu 2 (OH) 3 Cl shows a relatively uniform morphology of nanocuboids ( Figure S2).
Furthermore, we discovered that the STAM-17-OEt crystal shape is related to the morphology of Cu 2 (OH) 3 Cl. Using these irregularly sized Cu 2 (OH) 3 Cl to react with 5-ethoxyisophthalic acid resulted in irregularly sized STAM-17-OEt crystals ( Figure S3). STAM-17-OEt crystals with regular microslate morphology have been selected for amino acid modification to avoid performance differences caused by irregular sizes. i o (A m -2 ) α a α c C R C O P (V) 1 0.5 0.5 1 1000 0.5 In Table S4, i o is the exchange current density, and α a and α c are the anode and cathode transfer coefficients, respectively. C R and C O are reduction and oxidation species constants, respectively. P is the external potential. By adjusting the values of C O and P, the dimensionless current densities at different overpotentials can be displayed. S-44/S-44